Light-Guided Nanomotor Systems for Autonomous Photothermal

Dec 27, 2017 - Machines have greatly contributed to the human civilization, enabling tasks beyond our capacities for improved quality of life. Recentl...
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Light-guided Nanomotor Systems for Autonomous Photothermal Cancer Therapy Hyunsik Choi, Geon-Hui Lee, Ki Su Kim, and Sei Kwang Hahn ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16595 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Light-guided Nanomotor Systems for Autonomous Photothermal Cancer Therapy

Hyunsik Choi,† Geon-Hui Lee,† Ki Su Kim,#,§,* and Sei Kwang Hahn†,§,*

† Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Korea. # Department of Organic Materials Science and Engineering, College of Engineering, Pusan National University, 2 Busandaehak-ro 63 beon-gil, Gumjeong-gu, Busan 46241, Korea § PHI BIOMED Co., #613, 12 Gangnam-daero 65-gil, Seocho-gu, Seoul 06612, Korea.

* CORRESPONDING AUTHOR FOOTNOTE Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail address: [email protected] (S. K. Hahn) Tel.: +82 51 510 2496; Fax: +82 51 512 8175; E-mail address: [email protected] (K.S. Kim)

[KEYWORDS] nanomotor; light responsive; motion control; chemotaxis; photothermal cancer therapy

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ABSTRACT Machines have greatly contributed to the human civilization, enabling tasks beyond our capacities for improved quality of life. Recently, the progress in nanotechnology has triggered to build a miniaturized machine of nano-scale. In this context, synthetic nanomotors have gained considerable interest because of their great promise for diverse applications. Currently, the movement control of these nanomotors has been widely investigated using various stimuli. Here, we demonstrate near-infrared (NIR) light controlled on/off motion of stomatocyte nanomotors powered by the conversion of hydrogen peroxide. The nanomotors encapsulating naphthalocyanine (NC) are aggregated or separated (collective motion) with or without NIR light illumination, resulting in the well-controlled movement. Remarkably, the nanomotors can move directionally toward hydrogen peroxide released from cancer cells and photothermally ablate the cancer cells. Taken together, our stomatocyte nanomotor systems can be effectively harnessed for autonomous photothermal cancer therapy.

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INTRODUCTION A variety of synthetic micro and nanomotors in the forms of Janus particles,1-4 rods,58

helix structures,9-11 and tubules12-15 have been developed as a biomimetic system of natural

molecular motors and micro-organisms. These synthetic motors have been widely investigated for drug delivery,16-20 diagnostics,21-23 micro surgery,24,25 analyte capturing26,27 and chemical detoxification.28-30 In addition, the recent movement control of these motors includes the state (propel and stop) and the direction of motion triggered by various stimuli such as ultrasound,5-7 electricity,31,32 magnetic field,10,33 heat,34-36 and fuel gradient.37,38 Among these stimuli, light can be one of the most powerful physical triggers for spatiotemporal control of the object motion especially in nanotechnology applications due to the facile on-demand on/off control. However, most of the light-responsive synthetic motors are based on heavy metals which do not provide a suitable soft interface for biological systems.3,12,14,39-42 Recently, only a few motor systems have been reported using polymeric bilayers and catalytic metals.43,44 The polymeric bilayer motor in a micrometer scale might be difficult to perform complicated and delicate tasks for biomedical applications. To circumvent these issues, Wilson et al. developed a stomatocyte nanomotor of bowlshaped polymersomes encapsulating catalytic platinum nanoparticles (Pt NPs). The polymersome was prepared with poly(ethylene glycol)-block-poly(styrene) (PEG-b-PS).45 The ‘engine’ of prepared Pt NPs was entrapped selectively within the nanocavity of the stomatocyte. Pt NPs have been used as a catalyst for the vigorous decomposition of hydrogen peroxide (H2O2, ‘fuel’). Dioxygen converted by the catalytic reaction of Pt NPs diffuses through the narrow opening of the bowl-shaped structure of ‘nozzle’, which in turn

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produces mechanical energy by jet propulsion. Using this stomatocyte nanomotor, they demonstrated the autonomous movement towards H2O2 secreting cells by chemotaxis46 and drug delivery.47 In addition, they successfully performed the reversable motion control by the thermally responsive valve and brake.48 The opening of stomatocytes was widened or narrowed by the temperature change, thus controlling the access degree to H2O2. However, the synthetic process of this nanomotor with valve/brake was relatively complicated without the motion control in response to stimuli. However, the nanomotor was synthesized by the relatively complicated process and the speed of only each nanomotor was regulated without cooperating one another.49 It might not be suitable for biomedical applications including chemical sensing and cancer therapy. Thus, the collective motion (aggregation and separation) control of nanomotors in a simple responsive manner is highly required for biomedical applications such as biosensing and cancer therapy. Here, we have developed a NIR light triggered nanomotor system for active photothermal cancer therapy using PEG44-b-PS141 copolymers. Since the PEG in the copolymer is dehydrated above 55°C, PEG44-b-PS141 chain becomes hydrophobic,50 causing the collective motion of stomatocyte nanomotors. Thus, we simply prepared PEG44-b-PS141 nanomotors encapsulating Cu (II) 5,9,14,18,23,27,32,36-octabutoxy-2,3naphthalocyanine (NC) and Pt NPs. NC can be used as a strong NIR light absorber, and Pt NPs as an engine and catalytic decomposer of H2O2. The NIR light illumination could trigger temperature responsive behaviors of stomatocyte nanomotors. In addition, the nanomotors autonomously moved toward H2O2 released from cancer cells by chemotaxis and showed photothermal ablation effect on the cancer cells under NIR light illumination, demonstrating the feasibility for photothermal cancer therapy.

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MATERIALS AND METHODS Materials. Copper bromide (CuBr), N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA), styrene, potassium tetrachloroplatinate (II) (K2PtCl4), poly(vinyl pyrrolidone) (PVP, Mn = ca. 10,000), ascorbic acid, 1,4-dioxane, Krebs-Ringer bicarbonate (KRPG) buffer and sodium azide were purchased from Sigma Aldrich (St. Louis, MO). PEG macroinitiator (Mn = 1000 g/mol) was obtained from Iris Biotech GmbH (Germany). Copper (II) 5,9,14,18,23,27,32,36-octabutoxy-2,3naphthalocyanine (NC) was purchased from Tokyo Chemical Industry (Japan). Tetrahydrofuran (THF), methylene chloride (MC), methanol (MeOH), and anisole were obtained from Samchun Pure Chemicals (Korea). Chloroform-d was purchased from Cambridge Isotope (Cambridge, MA). Chlorin e6 was purchased from Santa Cruz Biotechnology (Dallas, TX). Synthesis of PEG41-b-PS183 copolymers. PEG-b-PS copolymers were synthesized as reported elsewhere with a slight modification.51 In brief, CuBr (22 mg) was placed into a Schlenk flask with a magnetic stirring bar. PMDETA (26 mg) in anisole (0.5 mL) and styrene (4.8 g) in anisole (4 mL) were added via a syringe. After cooling on the ice bath, PEG macroinitiator (100 mg) in anisole (0.5 mL) was added via a syringe followed by degassing with Ar in the reaction solution. Then, the Schlenk flask was placed into an oil bath preheated to 90ºC to initiate the synthesis of PEG-b-PS. The reaction process was monitored by proton nuclear magnetic resonance (1H NMR, Bruker, Germany) at the predetermined time point. When the required molecular weight was achieved, the reaction was terminated by cooling to room temperature after stirring for 2 h. The resulting solution was diluted in excess MC and filtered through a basic alumina column to remove the remaining Cu catalyst. The PEG-b-PS copolymer was obtained by precipitation in MeOH and dried under vacuum overnight. The PEG-b-PS copolymer was characterized by 1H NMR in CDCl3.

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Preparation of PVP capped Pt NPs. PVP-capped Pt NPs were synthesized as previously reported elsewhere.46 In brief, K2PtCl4 aqueous solution (20 mM, 4 mL) was added into 40 mg of PVP, followed by stirring for 48 h. After that, 35 mg of ascorbic acid in 1 mL of DI water was added into the solution. The resulting solution was sonicated in a bath sonicator (Powersonic 410, Hwasin Tech, Korea) at room temperature for 1 h. Then, the solution was centrifuged at 15,000 rpm for 15 min using an ultracentrifuge (Supra 30K, Hanil Science Industrial Co., Korea). The prepared Pt NPs were characterized by dynamic light scattering (DLS, NANO-ZS, Malvern, UK). We repeated the preparation of Pt NPs thrice to maximize the quantity of Pt NPs encapsulated in the stomatocyte. Self-assembly and characterization of stomatocytes encapsulating NC and Pt NPs. PEG-b-PS (10 mg) and NC (0.5 mg) were fully dissolved in a mixture of THF/1,4-dioxane (4/1, v/v, 1 mL). DI water (0.3 mL) was slowly added into the solution with a syringe pump at a rate of 1 mLh-1 and then the prepared aqueous solution of Pt NPs with high concentration (0.7 mL) was added to the solution. Stomatocytes encapsulating NC and Pt NPs were obtained after extensive dialysis against DI water for at least 48 h. The size and morphology of the nanomotors were analyzed by energy-dispersive X-ray (EDX) equipped high resolution - transmission electron microscopy (HRTEM, JEM-2200FS, JEOL, Japan) at 200 kV. In addition, the encapsulation efficiency of NC was determined with a UV-Vis spectrophotometer (s-3100, Scinco, Korea). In vitro release test of NC from stomatocyte nanomotors. In vitro release tests of NC from stomatocyte nanomotors were performed over 24 h using a pre-washed dialysis tube (MWCO 10 kDa). The dialysis tube contained 2 mL of the NC loaded nanomotors aqueous solution and the dialysis tubes were incubated in 10 mL of PBS at pH 7.4 and 37 °C for a day. At pre-determined time intervals, samples of the incubation medium were collected and the whole medium was

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replaced with 10 mL of fresh PBS. The amount of NC in the collected samples was quantified by UV-vis spectrophotometer. Three replicates were performed for the in vitro release tests. Particle size and movement analysis of stomatocyte nanomotors. The nanomotor solution (100 μL) was added into 96-well plates. A portable NIR laser (808 nm, Jet Lasers Photonics, Shenzhen, China) with a power density of 2 W/cm2 was illuminated onto the well for 10 min. The temperature was measured per 2 min followed by fast cooling in an ice bath to 37C (2 cycles). The aggregation and separation of nanomotors were observed by optical microscopy (TCS SP5, Leica, Germany) with particle size measurement by Nanosight (NS500, Malvern). In addition, the diffusion coefficient of the particles was determined by DLS software. For movement analysis of nanomotors, time lapse optical images were taken for 9 s with 1 frames per 180 ms. The tracking path and velocity of nanomotors was analyzed with ImageJ. Fluorescence imaging for cellular uptake of nanomotors. PEG-b-PS (10 mg) and Chlorin e6 (0.5 mg) were fully dissolved in a mixture of THF/1,4-dioxane (4/1, v/v, 1 mL). DI water (0.3 mL) was slowly added into the solution with a syringe pump at a rate of 1 mLh-1 and then the prepared aqueous solution of Pt NPs (0.7 mL) was added to the solution. Chlorin e6 loaded nanomotors were obtained after extensive dialysis against DI water for at least 48 h. Meanwhile, MDA-MB 231 cells were seeded on an eight-chamber glass slide at a density of 5 × 104 cells/well and cultured in DMEM supplemented with 10 vol% of FBS and 1 wt% of antibiotics in a humidified 5% CO2 incubator at 37 °C. The culture medium was replaced with FBS-free DMEM. Chlorin e6 loaded nanomotors were added to the wells of culture slides. The cells were incubated for 4 h, washed with PBS three times, and fixed with 4% paraformaldehyde in PBS. After DAPI staining for 10 min, the cells were observed with a confocal microscope (FV1000, Olympus America Inc.).

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Assessment of nanomotor chemotaxis to cancer cells. To measure the amount of H2O2 from cancer cells, MDA-MB 231 human breast cancer cells were seeded in complete growth medium (l08 cellsmL-1) at 96-well plates (0.1 mLwell-1) and allowed to adhere overnight. The cells were then washed with warm KRPG twice and the amount of H2O2 was measured by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (ThermoFisher Scientific, Waltham, MA) in the presence of 1 mM sodium azide to prevent the enzymatic degradation of H2O2. To evaluate chemotaxis of nanomotors, the cells were incubated with KRPG buffer for 4 h and transferred into the transwell insert filters (6.5 mm diameter inserts, 3 µm pore size, Corning Life Sciences, Corning, NY) after placing Pt NP loaded stomatocytes in KRPG solution into the 24-well. Time lapse optical images were taken for 9 s with 1 frames per 180 ms. The tracking of nanomotors was analyzed with ImageJ. The average distance of nanomotors travelled in the minimal time interval was obtained as the velocity of nanomotors. In vitro photothermal ablation of cancer cells. MDA-MB 231 cells at a density of 106 cellsmL1

were seeded on 96-well plates and 50 µL of the nanomotor aqueous solution was added with

different concentrations of 0, 25, 50,100, 200 µgmL-1. Then, the mixture of cells and nanomotors was exposed by portable NIR laser with a power density of 2 Wcm-2 for 10 min. Finally, the cells were analyzed by trypan blue exclusion assay and MTT assay. Statistical analysis. Statistical analysis was carried out via the t-test using the software of SigmaPlot10.0 (Systat Software Inc. San Jose, CA). The values for **P < 0.01 was considered statistically significant. Data are expressed as means ± standard deviation (SD) from several separate experiments. All experiments were performed triplicate and 30 nanomotors were measured for each group.

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RESULTS AND DISCUSSIONS As schematically shown in Figure 1, stomatocyte nanomotors autonomously move by catalytic degradation of H2O2 at 37°C (on state). When the NIR light is illuminated to the nanomotors, the entrapped NC molecules absorb NIR light and the membrane temperature increases gradually. Around 55°C, the nanomotors become aggregated one another due to the enhanced hydrophobicity of PEG and stop the mechanical movement spontaneously (off state). This phenomenon is reversible. Upon cooling, the separation occurs and the speed of nanomotors is recovered due to the rehydration of PEG blocks (on state). For the preparation of stomatocyte nanomotors, PEG44-b-PS141 copolymer was synthesized via atom transfer radical polymerization (ATRP). The successful synthesis was confirmed by 1

H NMR (Supporting Information, Figure S1). After that, block copolymers and NC were

dissolved in the mixture of tetrahydrofuran (THF) and dioxane (4/1, v/v). Distilled water was slowly added into the solution at a rate of 1 mLh-1 to form the self-assembled stomatocytes. Meanwhile, poly(vinyl pyrrolidone) (PVP) coated Pt NPs were prepared to enhance the hydrophilicity and the encapsulation efficiency for the catalytic motion. The hydrodynamic size of Pt NPs was ca. 20 nm (PDI: 0.235) according to the dynamic light scattering (DLS, Figure S2A). The Pt NPs were added into the organic solution of copolymers to capture nanoparticles into the outer cavity of stomatocytes. After dialysis against an excess amount of distilled water for 48 h, stomatocytes containing NC and Pt NPs were obtained by removing the organic solvent in the mixed solution. The characteristic morphology of stomatocytes in a bowl-shape was confirmed by high resolution - transmission electron microscopy (HR-TEM). The particle size of each stomatocyte was ca. 150 nm and the opening size was ca. 10 nm. The Pt NPs were

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entrapped in the outer cavity of stomatocytes (Figure 2A). The number of entrapped Pt NPs was 10 ~ 20 at the highly concentrated Pt NPs solution (Figure 2SB). In order to confirm the successful encapsulation of NC and Pt NPs, we analysed Pt and copper (Cu), the main elements of Pt NPs and NC in the stomatocyte, respectively. Energy-dispersive X-ray spectroscopy (EDX) mapping revealed the incorporation of both Pt and Cu in the stomatocyte (Figure 2B and C). UV-Vis-NIR spectroscopy also corroborated the encapsulation of NC, exhibiting strong NIR absorption band from 750 nm to 850 nm (Figure S3A). Then, the encapsulation efficiency of NC was calculated by the comparison of absorbance at 850 nm between free NC and NC in the stomatocyte. The encapsulation efficiency of NC was almost 100 % (inset of Figure. S3B). However, the entrapped NC was hardly released from the stomatocyte within 24 h (Figure. S3B) because of the strong hydrophobic characteristics of NC molecules. After confirming the successful preparation of stomatocyte nanomotors containing Pt NPs and NC, the motion of the nanomotors was characterized by nanoparticle tracking analysis (NTA). In contrast to DLS which provides the ensemble size distribution, NTA can analyse the individual nanomotor by recording the real-time movement of nanomotors. 52

The concentration of nanomotors measured by NTA was 3  108 mL-1 at physiological

temperature of 37°C. For the motion analysis, a video was recorded for 40 s at the rate of 25 framess-1. Without the fuel (H2O2), stomatocyte nanomotors moved randomly by Brownian motion and did not show any directionality (Figure 2D and Figure S4A). However, after 20 µL of H2O2 solution as a fuel was added to 2 mL of nanomotor solution (0.03 %, v/v), the nanomotors diffuse faster than control group (Figure 2E and Figure S4B). In addition, the diffusion coefficient of the particles can be determined by DLS software. The

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diffusion coefficient of the nanomotor increased 4-folds from 1.1 µm2s-1 to 4.3 µm2s-1 (Figure 2F). Moreover, due to the significant increase of diffusion, their ‘apparent’ size was smaller than that of the same structures without the fuel (Figure 2G).45 In order to confirm the on/off motion of nanomotors triggered by NIR light illumination, the temperature responsive behaviour of stomatocytes was investigated as shown in Figure 3. As shown in Figures 3A and B, the initial average hydrodynamic diameter of the nanomotors was ca. 220 nm at 37°C. After illuminating 808 nm laser, the temperature increased from 37°C to 55°C within 10 min. Around 55°C, the size of nanomotors became rapidly bigger than 5400 nm in the form of aggregates. However, when the NIR laser was turned off, cooling led to the separation of the nanomotor aggregates to the individual nanomotor, due to the rehydration of PEG blocks. Since the temperature responsive behaviour of stomatocyte nanomotor is reversible, the aggregation and separation are switchable, and can be repeated with multiple cycles by the facile lighttriggered temperature change. In addition, optical microscope images clearly showed separation (Figure 3C), aggregation (Figure 3D), semi-separation (Figure 3E) and re-separation state (Figure 3F) upon NIR light illumination. Based on the temperature responsive behaviours, we investigated the NIR lighttriggered “on/off” motion change of stomatocyte nanomotor systems (Figure 4). When NIR light was illuminated and the temperature increased to 55°C, the nanomotors were aggregated together, reducing the speed compared to individual nanomotors (Figure S4C). In contrast, after cooling, the movement of individual nanomotor was recovered as same as that before the heating state (Figures 4A and B). The velocity of nanomotors were measured from tracking path for 9 s. As shown in Figure 4C, the diffusion process was switchable

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and repeatable. The velocity decreased from 33.6 µms-1 to 3.5 µms-1 by NIR light illumination (“light on”). In addition, the change of nanomotor speed was measured with increasing H2O2 concentration (0, 0.0003, 0.003, 0.03 %, v/v) (Figure 4D). Without NIR light illumination, the velocity of nanomotors increased with increasing H2O2 concentration at 37°C. Although the velocity at 0.0003 % of H2O2 was similar to that without H2O2, they moved directionally and autonomously as shown in Figure 2D. Under NIR light illumination, the change of velocity was very small and the aggregation of nanomotors was independent of the fuel concentration. The cancelling net propulsion by NIR light illumination might be caused by the random aggregation of nanomotors or the closure of the stomatocyte opening during the aggregation. Since the central part of aggregates has less chance to meet the fuels (H2O2), the consumption of fuels can be controlled for the mechanical energy. After confirming NIR light-triggered “on/off” motion control of nanomotors, we evaluated the chemotaxis of nanomotors to cancer cells (MDA-MB 231). Since cancer cells release H2O2,53 we assume our nanomotor can move toward the tumor tissues. First, the release of H2O2 from cancer cells was assessed by the Amplex red assay. MDA-MB 231 cells constitutively released enough H2O2 to attract nanomotors. The cumulative amount at 4 h (6.5 µM per 106 cell) was comparable to the amount of H2O2 produced by activated neutrophils (Figure S5). To evaluate chemotaxis of nanomotors, 100 µL of transferred cells (108 mL-1) in insert filters and empty filters (control group) were embedded into the transwell. After stabilizing fluid flow, the movement of nanmotors was investigated in steady state (Figures 5A and B). When Pt NPs loaded nanomotors were placed with cancer cells, the nanomotors autonomously moved toward the cancer cells at a velocity of 8.97 µms-1 (Figures 5D and E). However, there was no such phenomenon observed in

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the control group (Figure 5C). In addition, when the nanomotors were incubated with cancer cells and H2O2 scavenger, as we expected, the nanomotors did not show the chemotaxis behavior but the random walk (Figure S6A). We calculated the average X displacement of 30 nanomotors for 9 s. There was no statistically significant difference in the average displacement between those without cancer cells and with cancer cells after H2O2 scavenging. However, in the cancer cell group, the nanomotors obviously moved toward cancer cells (Figure S6B). Without cancer cells, the nanomotors just randomly moved at a velocity of 6.54 µms-1 (Figure 5D). This velocity of nanomotors with and without cancer cells was consistent with the results of 0.0003 % H2O2 (about 98 µM) and without H2O2 (0 %) in Figure 4D. Before in vitro photothermal test, we conducted confocal imaging to investigate the cellular uptake of nanomotors (Figure S7). After 4 h incubation, nanomotors were well-distributed in the cytosol of MDA-MB 231 cells confirming the effective cellular uptake of the nanomotors by non-specific endocytosis.54 Finally, we investigated the photothermal effect of stomatocyte nanomotors using MDA-MB 231 cells. Before NIR light illumination, most of the cells were alive with intact cell membranes, not being stained by the trypan blue solution (Figure 5G). However, a lot of cells were stained by trypan blue after NIR light illumination, reflecting the photothermal ablation effect of nanomotors on cancer cells (Figure 5H). MTT assay also showed the similar results with the trypan blue staining data (Figure 5F). There was no cytotoxicity up to the high level concentration of 200 µgmL-1 without NIR light illumination. However, nanomotors showed the significant cytotoxicity with NIR light illumination.

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CONCLUSIONS A NIR light triggered polymeric stomatocyte nanomotor was successfully developed for active photothermal cancer therapy. The stomatocyte nanomotor was prepared with PEG44-b-PS141 encapsulating Pt NPs and NC. The nanomotor autonomously moved by the Pt-catalysed degradation of H2O2. Remarkably, NIR light illumination triggered the conformational change of PEG44-b-PS141 due to the temperature increase of NC in the membrane, resulting in reversible aggregation around 55°C. Furthermore, the nanomotors showed directional movement toward cancer cells and photothermal ablation effect on the cancer cells. Taken together, this nanomotor system might be used to pave the way to the effective photothermal cancer therapy.

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AUTHOR INFORMATION * Corresponding Author Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail address: [email protected] (S. K. Hahn) Tel.: +82 51 510 2496; Fax: +82 51 512 8175; E-mail address: [email protected] (K.S. Kim)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Nano·Material Technology Development Program (No. 2017M3A7B8065278) and the Basic Science Research Program (2017R1E1A1A03070458) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea. This work was also supported by the World Class 300 Project (R&D) (S2482887) of the Small and Medium Business Administration (SMBA), Korea.

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26. Uygun, M.; Singh, V. V.; Kaufmann, K; Uygun D.A.; de Oliveira, S.D.; Wang, J. Micromotor-Based Biomimetic Carbon Dioxide Sequestration: Towards Mobile Microscrubbers. Angew. Chem. Int. Ed. 2015, 54, 12900-12904. 27. Magdanz, V.; Guix, M.; Hebenstreit, F.; Schmidt O. G. Dynamic Polymeric Microtubes for the Remote-Controlled Capture, Guidance, and Release of Sperm Cells. Adv. Mater. 2016, 28, 4084-4089. 28. Singh, V. V.; Sánchez, B. J.; Sattayasamitsathit, S.; Orozco, J.; Li, J.; Galarnyk, M.; Fedorak Y.; Wang J. Multifunctional Silver-Exchanged Zeolite Micromotors for Catalytic Detoxification of Chemical and Biological Threats. Adv. Funct. Mater. 2015, 25, 2147-2155. 29. Sánchez, B. J.; Sattayasamitsathit, S.; Gao, W.; Santos, L.; Fedorak, Y.; Singh, V. V.; Orozco, J.; Galarnyk, M.; Wang J. Self-Propelled Activated Carbon Janus Micromotors for Efficient Water Purification. Small 2015, 11, 499-506. 30. Wu, Z.; Li, T.; Gao, W.; Xu, T.; Sánchez, B. J.; Li, J.; Gao, W.; He, Q.; Zhang, L.; Wang,

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33. Tottori, S.; Zhang, L.; Qiu, F.; Krawczyk, K. K.; Obregón, A. F.; Nelson B. J. Magnetic Helical Micromachines: Fabrication, Controlled Swimming, and Cargo Transport. Adv. Mater. 2012, 24, 811-816. 34. Balasubramanian, S.; Kagan, D.; Manesh, K. M.; CalvoMarzal, P.; Flechsig, G.-U.; Wang, J. Thermal Modulation of Nanomotor Movement. Small 2009, 5, 1569–1574. 35. Sanchez, S.; Ananth, A. N.; Fomin, V. M.; Viehrig, M.; Schmidt, O. G. Superfast Motion of Catalytic Micromotors at Physiological Temperature. J. Am. Chem. Soc. 2011, 133, 14860–14863. 36. Magdanz, V.; Stoychev, G.; Ionov, L.; Sanchez, S.; Schmidt, O. G. Stimuli-Responsive Microjets with Reconfigurable Shape. Angew. Chem., Int. Ed. 2014, 126, 2711–2715. 37. Hong, Y.; Blackman, N.; Kopp, N.; Sen, A.; Velegol, D. Chemotaxis of Nonbiological Colloidal Rods. Phys. Rev. Lett. 2007, 99, 178103. 38. Wilson, D. A.; Nijs, B.; de Blaaderen, A.; van Nolte, R. J. M.; van Hest, J. C. M. Fuel Concentration Dependent Movement of Supramolecular Catalytic Nanomotors. Nanoscale 2013, 5, 1315–1318. 39. Li, Y.; Mou, F.; Chen, C.; You, M.; Yin, Y.; Xu, L.; Guan, J. Light-Controlled Bubble Propulsion of Amorphous TiO2/Au Janus Micromotors. RSC Adv. 2016, 6, 1069710703. 40. Zhang, Q.; Dong, R.; Wu, Y.; Gao W.; He, Z.; Ren, B. Light-Driven Au-WO3@C Janus Micromotors for Rapid Photodegradation of Dye Pollutants. ACS Appl. Mater. Interfaces 2017, 9, 4674-4683.

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41. Jang, B.; Hong, A.; Kang, H. E.; Alcantara, C.; Charreyron, S.; Mushtaq, F.; Pellicer, E.; Bü chel, R.; Sort, J.; Lee, S. S.; Nelson, B. J.; Pane, S. Multiwavelength LightResponsive Au/B-TiO2 Janus Micromotors. ACS nano 2017, 11, 6146-6154. 42. Gao, Y.; Mou, F.; Feng, Y.; Che, S.; Li, W.; Xu, L.; Guan, J. Dynamic Colloidal Molecules Maneuvered by Light-Controlled Janus Micromotors ACS Appl. Mater. Interfaces 2017, 9, 22704-22712. 43. Gao, W.; Sattayasamitsathit, S.; Orozco, J.; Wang, J. Highly Efficient Catalytic Microengines: Template Electrosynthesis of Polyaniline/Platinum Microtubes. J. Am. Chem. Soc. 2011, 133, 11862-11864. 44. Gao, W.; Sattayasamitsathit, S.; Uygun, A.; Pei, A.; Ponedal, A.; Wang, J. Polymerbased Tubular Microbots: Role of Composition and Preparation. Nanoscale, 2012, 4, 2447-2453. 45. Wilson, D. A.; Nolte, R. J. M.; van Hest, J. C. M. Autonomous Movement of PlatinumLoaded Stomatocytes. Nature Chemistry 2012, 4, 268-274. 46. Peng, F.; Tu, Y.; van Hest, J. C. M.; Wilson, D. A. Self-Guided Supramolecular CargoLoaded Nanomotors with Chemotactic Behavior towards Cells. Angew. Chem. Int. Ed. 2015, 54, 11828-11831. 47. Tu, Y.; Peng, F.; Andre A. A. M.; Men, Y.; Srinivas, M.; Wilson, D. A. Biodegradable Hybrid Stomatocyte rs for Drug Delivery. ACS Nano 2017, 11, 1957−1963. 48. Tu,Y.; Peng, F.; Sui, X.; Men, Y.; White, P. B.; van Hest, J. C. M.; Wilson, D. A. SelfPropelled

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49. Duan, W.; Liu, R.; Sen, A. Transition between Collective Behaviors of Micromotors in Response to Different Stimuli. J. Am. Chem. Soc. 2013, 135, 1280-1283. 50. Hocine, S.; Cui, D.; Rager, M. N.; Cicco, A. D.; Liu, J. M.; Bakala, J. W.; Brûlet, A.; Li, M. H. Polymersomes With PEG Corona: Structural Changes and Controlled Release Induced by Temperature Variation. Langmuir 2013, 29, 1356-1369. 51. Kim, K. T.; Cornelissen, J. J. L. M.; Nolte, R. J. M.; Van Hest, J. C. M. A Polymersome Nanoreactor with Controllable Permeability Induced by Stimuli-Responsive Block Copolymers. Adv. Mater. 2009, 21, 2787-2791. 52. Filipe, V.; Hawe, A.; Jiskoot, W. Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates. Pharm. Res. 2010, 27, 796-810. 53. Szatrowski, T. P.; Nathan, C. F. Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells. Cancer Research 1991, 51, 794-798. 54. Mao, Z.; Zhou, X.; Gao, C. Influence of Structure and Properties of Colloidal Biomaterials on Cellular Uptake and Cell Functions. Biomater. Sci. 2013, 1, 896-911.

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FIGURES

A Self-propelling by decomposition of H2O2

Dehydration of PEG upon heating

Hydrophilic

Hydrophilic

Hydrophobic

Light on

Light off

T

T

Move

Stop / photothermal effect

Move : PEG-b-PS

B

Rehydration of PEG upon cooling

Autonomous tumor targeting (chemotaxis)

: Pt NP

: Naphthalocyanine

Stop & photothermal ablation

: H2O2

: ½ O2 + H2O

Cancer cell death

Hydrogen peroxide gradient

Figure 1. (A) Schematic representation of a light-guided nanomotor system using PEG44b-PS141 / naphthalocyanine (NC) and Pt nanoparticles (Pt NPs) powered by the conversion of hydrogen peroxide (H2O2). (B) Schematic illustration for the autonomous photothermal ablation of cancer cells using the nanomotors.

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ACS Applied Materials & Interfaces

A

B

C Cu

Pt

D

E 3

Y (μm)

Y (μm)

3

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G Concentration percent (%)

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-1

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20 w/o H2O2 w/ H2O2 15

10

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0 0

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200

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Figure 2. (A) HR-TEM image of nanomotors and the corresponding EDX map of (B) Pt (green) and (C) Cu (red). The trajectories of 3 representative nanomotors (particle concentration = 3  108 mL-1) by nanoparticle tracking analysis for 3 s (D) without and (E) with H2O2 (0.03%, v/v). (F) The diffusion coefficient change of nanomotors depending on the presence of H2O2 (37 °C). (G) The size distribution of nanomotors before (black) and after (blue) the addition of H2O2. **P ≤ 0.01 with H2O2 vs without H2O2 (n = 3, scale bar = 20 nm).

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A

B 60

8

Light On Off 55

Size (μm)

50 45

Off

On

4

2

40 35

On

6

o

Temperature ( C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Light On Off 0

10

On

On

Off

20

30

0 40

0

10

20

30

40

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Time (min)

C

D

E

F

Figure 3. (A) The cycles of temperature change per 2 min and (B) the size change of nanomotors per 2min under NIR light illumination. The optical images of nanomotors (C) before and (D) after NIR light illumination, and (E) during and (F) after cooling (scale bar = 20 µm).

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ACS Applied Materials & Interfaces

A

B 3

Y ( μm)

Y (μm)

3

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0 0

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.

**

. 40

30 20 10

30 20 10

0

0 Light On

Off

On

Off

0

0.0003

0.003

0.03

H2O2 concentration (% v/v)

Figure 4. The trajectories of 3 representative nanomotors (A) after NIR light illumination and (B) cooling with H2O2 (0.03 %, v/v). In case of (A), the center of aggregation was used for the trajectory analysis. (C) The velocity change of nanomotors according to NIR light illumination (0.03 %, v/v) and (D) various H2O2 concentrations (0 %, 0.003 % and 0.03 %, v/v). **P ≤ 0.01 vs 0 % H2O2 (n = 30 nanomotors for each group).

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A

B

KRPG buffer

Low

C

High

60

Y (μm)

Y (μm)

H2O2

D 60

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0 0

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.

6 4 2 0

100 80 60 40

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G

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Figure 5. Schematic illustration for the chemotaxis evaluation of nanomotors (A) without (control group) and (B) with MDA-MB 231 cells. The tracking path of 6 representative nanomotors (C) without and (D) with MDA-MB 231 cells. (E) The average velocity of 30 nanomotors corresponding to the tracking path in (C) and (D). (F) Effect of photothermal ablation of nanomotors on the viability of MDA-MB 231 cells. **P ≤ 0.01 vs without laser (n = 3). The photothermal effect of nanomotors by trypan blue exclusion assay (G) before and (H) after NIR light illumination. Red circles indicate the aggregate of nanomotors (scale bar = 100 µm).

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– For Table of Contents Use Only –

Light-guided Nanomotor System for Autonomous Photothermal Cancer Therapy

Hyunsik Choi,† Geon-Hui Lee,† Ki Su Kim,#,§,* and Sei Kwang Hahn†,§,*

Autonomous tumor targeting (chemotaxis)

Stop & photothermal ablation

Cancer cell death

Hydrogen peroxide gradient

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